Latchezar D. Todorov

Evidence for the differential release of the cotransmitters ATP and noradrenaline from sympathetic nerves of the guinea‐pig vas deferens.

1. Experiments were carried out to quantify the stimulation‐evoked overflow of catecholamines and purines (ATP, ADP, AMP and adenosine) from an in vitro sympathetic nerve‐smooth muscle preparation of the guinea‐pig vas deferens and from isolated bovine adrenal chromaffin cells. The superfused preparations were stimulated for 60 s with electrical field stimulation (EFS; vas deferens), dimethylphenylpiperazinium (chromaffin cells) or KCl (both preparations). 2. Samples of superfusate were taken at 10 s intervals during the 60 s stimulation period for analysis of purines by HPLC‐fluorescence detection and catecholamines by HPLC‐electrochemical detection. 3. The evoked overflow of catecholamines and purines from chromaffin cells occurred with the same time course and in a constant ratio of approximately 4:1 (catecholamine to purine). These findings are compatible with the release of catecholamines and purines from a homogeneous population of exocytotic vesicles in the chromaffin cells. 4. The evoked overflow of purines and noradrenaline (NA) from the vas deferens preparation differed from the pattern of overflow from chromaffin cells and there was also some temporal disparity in the overflow of the two cotransmitters. The evoked overflow of ATP exceeded that of NA. In addition, the overflow of NA was tonic while the overflow of ATP and the other purines was phasic. 5. The EFS‐evoked overflow of NA and the purines from the guniea‐pig vas deferens preparation was examined after treatment with the neuronal amine‐uptake inhibitors desipramine and cocaine, the alpha 1‐adrenoceptor agonist methoxamine, the alpha 1‐adrenoceptor antagonist prazosin, the alpha 2‐adrenoceptor antagonists idazoxan and yohimbine, the noradrenaline‐depleting drug reserpine and the adrenergic neuron‐blocking agent guanethidine. The results of these studies, together with an analysis of the metabolic degradation of extracellular ATP, indicated that the temporal disparity in the overflow of NA and ATP is unlikely to be due to differences in the clearance of the cotransmitters or to the release of purines from non‐neuronal sites. These results indicate that evoked overflow of the cotransmitters accurately reflects release from nerves. This pattern of release from nerves suggests that the two cotransmitters are released from two separate populations of exocytotic vesicles. 6. Superfusion of the vas deferens with exogenous epsilon‐ATP, a fluorescent derivative of ATP, revealed that there was essentially no metabolism of the nucleotide over 60 s unless the tissue was subjected to EFS. Upon EFS, there was a rapid and nearly complete degradation of ATP with a corresponding increase in ADP, AMP and adenosine. This indicates the presence of a nerve stimulation‐dependent metabolism of ATP.

Chapter 2 Modulation of purinergic neurotransmission

Publisher Summary This chapter highlights some recent developments in the understanding of ATP as a cotransmitter. Four main topics of purinergic research are emphasized in the chapter: the storage and release of ATP and its regulation, the structure and classification of P2-receptor subtypes, the postjunctional effector mechanisms by which ATP mediates its neurotransmitter actions, and the mechanism of inactivation of the neurotransmitter actions of ATP by ATPases. Recent studies indicate that there are more than one population of storage vesicles in the nerves, as the release of various cotransmitters varies over time and can be differentially modulated by drugs. The subclassification of P2 receptors has advanced in the past few years because of the use of molecular biology methods allowing the cloning and expression of 14 different subclasses of P2 receptors, seven P2X, and seven P2Y. Determination of the functional significance of various receptor subtypes would be helped by the development of selective agonists and antagonists.

Release of soluble nucleotidases: a novel mechanism for neurotransmitter inactivation?

The support of the NIH and American Heart Foundation to Prof. D. P. Westfall, Astra Charnwood and the Wellcome Trust (C. K., P. S.), the Caledonian Research Foundation (C. K.) and Carnegie Trust (P. S.) is gratefully acknowledged. We also thank the Scottish Hospital Endowments Research Trust for refurbishing the laboratories in which some of these experiments were performed.

DIRECT MEASUREMENT OF THE RELEASE OF ATP AND ITS MAJOR METABOLITES FROM THE NERVE FIBRES OF THE GUINEA-PIG TAENIA COLI

1. The release of adenosine triphosphate (ATP), adenosine diphosphate, adenosine monophosphate and adenosine from guinea‐pig taenia coli in response to electrical stimulation of intramural nerves was measured directly using high performance liquid chromatography separation and fluorometric detection.

TEMPORAL DISSOCIATION OF THE RELEASE OF THE SYMPATHETIC CO‐TRANSMITTERS ATP AND NORADRENALINE

Based partially on an analogy with adrenal chromaffin cells, it is a commonly held view that the sympathetic nerve co-transmitters ATP and noradrenaline (NA) are stored in and released from the same vesicles within the nerve varicosity (Sneddon & Westfall 1984; Stjarne 1989). If ATP and NA originate from the same vesicles then the ratio of released NA to purine should remain constant during stimulation and the time course of release should be similar for both transmitters. We have tested this hypothesis with the in vitro sympathetic nerve smooth muscle preparation of the guinea-pig vas deferens. The approach was to use a superfusion system that rapidly removed the released transmitter from the preparation at a constant rate. The preparations were stimulated for 1 min with transmural nerve stimulation at frequencies of 2 ,4 or 8 Hz. Samples of superfusate were taken at 10 s intervals for analysis of adenine nucleotides and adenosine by HPLC-fluorescence detection and catecholamines by HPLC-electrochemical detection (Sedaa et al. 1990). While what is actually quantified in this type of experiment is the amount of transmitter that overflows from the preparation, the conditions are such that overflow reflects the amount of transmitter that is released from the nerves. Figure 1 shows the time course of overflow of NA and ATP from the vas deferens evoked by transmural stimulation. For both transmitters the magnitude of overflow was increased as the frequency of stimulation increased. It is apparent, however, that the time course of overflow differed markedly for the two transmitters. The release of NA increased with increasing numbers of pulses when stimulated at 2 or 4 Hz. When stimulated at 8 Hz the release of NA reached a peak at 30 s and remained constant for the remainder of the 1 min stimulation period. At the completion of the stimulation the overflow of NA gradually returned toward prestimulation levels. The release of ATP, on the other hand, reached a peak much more quickly, by about 20 s, and then declined dramatically even though the stimulation continued for 1 min. We monitored the overflow not only of ATP but ADP, AMP and adenosine as well. While there were quantitative differences in the overflow of these nucleotides and nucleosides the pattern of release was qualitatively similar for all four. Therefore, the transient nature of the overflow of ATP cannot be accounted for by its metabolism to other purines because, if this were so, one would expect an increasing accumulation with time of the total pool of adenine nucleotides and nucleosides. The primary mechanism for removing NA from the synapse once it is released is re-uptake into sympathetic nerves (Graefe & Bonisch 1988). In order to determine whether neuronal uptake influences the pattern of the overflow of NA we carried out experi-